DESCRIPTION: The goal of this proposal is to develop a clinically feasible protocol for ultrasound (US) mediated gene delivery (UMGD) of factor VIII (FVIII) to treat hemophilia A (HemA). Current treatment for HemA patients involves costly and inconvenient repeated infusions of protein concentrations. In a recent clinical trial, adenoassoicated viral vector (AAV) mediated gene therapy has shown excellent promise for treating hemophilia B1-3. However the limitation of accommodating large size gene such as FVIII in the AAV vector, the immune responses to the vector2, 3 and associated transgene products4, and limitation of repeated treatment with pre-existing immunity to AAV vector5, 6 significantly hinder the development of an effective AAV-mediated gene therapy treatment for HemA. Previously we demonstrated that UMGD can significantly enhance reporter gene transfer into the mouse7-9 and rat livers10. This nonviral gene transfer strategy can bypass many obstacles encountered by viral gene therapy. Most significantly, we have recently achieved therapeutic levels of FVIII following UMGD into HemA mice11. In order to facilitate the eventual translation of these technologies into human application, many technical issues, including treatment procedures and protocols, appropriate MB volumes and types, and US parameters and instrumentation require exploration in large animal models. We have successfully developed prototype US systems including several unfocused and semi-focused transducers to treat large tissue volumes in canine12 and swine13. We have also developed several new neutral and cationic MBs to facilitate gene transfer14. The current proposal focuses on the development of a safe and clinically feasible ultrasound technology along with suitable surgery techniques to achieve efficient gene transfer in large animal models, leading to high levels of FVIII gene expression. First, we will explore the best US parameters that can enhance gene transfer efficiency with minimal tissue damage in mice. In addition, our data indicate that transgene is principally expressed by hepatocytes following UMGD into the liver. We propose to make liver-specific constructs carrying a high-expressing FVIII variant gene in a MIP plasmid to further increase and prolong FVIII gene expression in vivo, in order to achieve phenotypic correction in HemA mice. Next, we will improve our US technology and surgery techniques to optimize gene transfer efficiencies in large animal models. Importantly, we will develop minimally invasive interventional radiologic techniques to deliver plasmid DNA (pDNA)/MB mixture into the target liver lobe combined with transcutaneous US treatment procedures in pigs. Long-term experiments will be performed in normal dogs using our most effective FVIII plasmid and the optimized therapeutic US method in combination with immunomodulation to achieve persistent and high-level FVIII gene expression. If successful, this project will facilitate the phenotypic correction in the HemA dog model and the eventual translation of this novel technology into human application, and could change fundamentally the way HemA patients are treated, with better patient outcomes.